Three-dimensional (3D) cell cultures are to date the golden standard in biomedical research fields due to their enhanced biological functions as compared to conventional two-dimensional (2D) cultures. 3D cell spheroids as well as organoids are better suited to replicate tissue functions, which enables their use both as in vitro models for basic research and toxicology, as well as building blocks used in tissue/organ biofabrication approaches. Culturing 3D spheroids from bone-derived cells is an emerging technology for both disease modelling and drug screening applications. Bone tissue models are mainly limited by the implementation of sophisticated devices and procedures that can foster a tissue-specific 3D cell microenvironment along with a dynamic cultivation regime. In this study, we consequently developed, optimized and characterized an advanced perfused microfluidic platform to improve the reliability of 3D bone cell cultivation and to enhance aspects of bone tissue maturation in vitro. Moreover, fluid shear stress, generated by fluid flow inside the chamber, was used to mimic the dynamic cell environment in bone architecture and thus improve an osteogenic 3D microenvironment in the multifunctional spheroid-array platform. The 3D cell cultures cultivated in our bone on a chip model exhibited increased mineralization and viability compare to static conditions. Thus, this system represents a powerful tool how to study bone physiology and pathophysiology in vitro.